Our long-range goal is to examine catalytic activities of core structures derived from class I and II aminoacyl- tRNA synthetases (aaRS), to test experimentally the hypothesis that protein synthesis began using two low- specificity amino acid activating enzymes coded by opposite strands of the same gene, and whose contemporary progeny are the ten class I and ten class II aaRS. Previous work on transfer RNA domains showed that acceptor stem minihelices can be specifically aminoacylated at rates within three orders of magnitude of those observed for the full-length tRNAs and thereby established the modularity of tRNA evolution. We have reexamined class I aaRS tertiary structures in the light of sequence entropies in multiple sequence alignments of approximately 1900 for each class. A new mosaic structure of the class I superfamily obtained in this manner reveals a core fragment whose sequences derive from discontinuous fragments of the N- and C- terminal b-a-b crossover connections from the Rossmann dinucleotide-binding fold, together with the amino acid specificity-determining helix from the core catalytic domain. This core structure is both modular and closely superimposible in all ten families of class I aaRS. We have demonstrated that a "minimal catalytic module", derived from TrpRS using protein design methods in collaboration with Brian Kuhlman, is quite active. Our first goal is to characterize this activity more fully for class I aaRS minimal catalytic domains, using steady state kinetics, active site mutation, and to evaluate the functional contributions of subsequently accumulated modules by constructing combinations of the minimal catalytic domains with other modular components from the mosaic hierarchy, notably the anticodon binding and CP1 insertion domains. Our second aim is to implement a similar strategy to examine catalytic activities derived from corresponding minimal catalytic domains from class II aaRS. Our goal is to demonstrate that active fragments of similar length can be derived from both aaRS classes as experimental support for the hypothesis. Our third aim is to adapt the protein design software used by Professor Kuhlman to simultaneously design pairs of class I and class II minimal catalytic domains that retain catalytic activity while improving their sense/antisense encoding. This research program promises to extend understanding not only of an important event in the origin of protein synthesis, but also constraints involved in sense/antisense coding of protein structures.